The overall objective of this project is to elucidate the physiological, and pharmacological significance and biochemical regulation of the two molecular forms of Na+,K+ -ATPase in the ferret heart. We have demonstrated that two molecular forms of Na+,K+-ATPase exist in ferret myocardial cells in approximately equal abundance; the high (alpha(alpha(+)) and the low (alpha) molecular with forms correspond to the high and low affinity ouabain binding sites, respectively. Inhibition of the isoenzymes by ouabain caused biphasic positive inotropic effects indicating that both of the alpha subunits are the pharmacological receptors for the cardiac glycosides. Although isoenzymes of Na+,K+-ATPase are widely distributed in different cell types, the physiological significance of having two isoforms of the Na pump in the cardiac cells is unknown. Ferret heart offers a unique opportunity for these studies. In the proposed study, the following experiments will be performed: 1) Possible physiological significance of the two isoforms of the Na pump will be examined. The hypothesis that alpha(+) and alpha forms of the Na pump operate at different rates and that insulin preferentially regulates the alpha(+) form of the Na pump will be tested. Insulin has been shown to stimulate the alpha(+) form of the Na pump of fat cells with no effect on the alpha form of the pump. 2) Relative abundance of the alpha(+) form increases during neonatal and early postnatal development. The hypothesis that these changes are closely related to changes in pharmacological effects of the glycoside and that a low affinity "neonatal" form is common among different species during early development will be tested. 3) The hypothesis that differences in affinity of the alpha subunits for the glycoside results from differences in turnover rate and/or stability of the phosphoenzymes will be examined. 4) The hypothesis that myocardial hypertrophy alters isoenzyme profiles and therefore glycoside sensitivity of the heart will be tested. 5) Thyroid hormone regulates a wide range of physiological functions including the expression of myosin isoenzymes and the total number of Na+,K+-ATPase units. Therefore, the hypothesis that thyroid hormone regulates the expression of the two isoforms of alpha subunits will be tested. Results of these studies will indicate if the relative activity or the relative abundance of two isoforms of Na+,K+-ATPase is under hormonal control or is affected by myocardial hypertrophy, and these changes are associated with alterations in sensitivity to the digitalis glycosides.